Pharmacokinetics and Bioavailability of the Isoflavones Formononetin

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Pharmacokinetics and Bioavailability of the Isoflavones Formononetin and Ononin and their In Vitro Absorption in Ussing Chamber and Caco-2 Cell Models Li-Yu Luo, Miao-Xuan Fan, Hai-Yu Zhao, Mingxing Li, Xu Wu, and Wenyuan Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00035 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Journal of Agricultural and Food Chemistry

Pharmacokinetics and Bioavailability of the Isoflavones Formononetin and Ononin and their In Vitro Absorption in Ussing Chamber and Caco-2 Cell Models Li-Yu Luo,† Miao-Xuan Fan,‡ Hai-Yu Zhao,# Ming-Xing Li,§ Xu Wu,§,* and Wen-Yuan Gao†,* †

School of Pharmaceutical Science and Technology, Tianjin University, Tianjin,

China ‡

Beijing Key Laboratory of Analysis and Evaluation on Chinese Medicine, Beijing

Institute of Drug Control, Beijing 102206, China # Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China §

Laboratory of Molecular Pharmacology, Department of Pharmacology, School of

Pharmacy, Southwest Medical University, Luzhou, Sichuan, China

*Corresponding Author: Telephone: +86-13920837932; E-mail: [email protected] (W.Y. Gao). Telephone: +86-13882770623; E-mail: [email protected] (X. Wu).

ACS Paragon Plus Environment


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ABSTRACT

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Formononetin and its glycoside ononin are bioactive isoflavones widely present in

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legumes. The present study investigated the pharmacokinetics, bioavailability and the

4

in vitro absorption of formononetin and ononin. After an oral administration to rats,

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formononetin showed a higher systemic exposure over ononin. The oral

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bioavailability of formononetin and ononin were 21.8% and 7.3%, respectively.

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Ononin was more bioavailable than perceived, and its bioavailability reached 21.7%

8

when its metabolite formononetin was taken into account. Both formononetin and

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ononin exhibited better absorption in large intestine segments than that in small

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intestine segments. Formononetin displayed a better permeability in all intestinal

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segments over ononin. Transport of formononetin across Caco-2 cell monolayer was

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mainly through passive diffusion, while ononin was actively pumped out by MRP2

13

but not P-gp. The results provide evidences for better understanding of the

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pharmacological actions of formononetin and ononin, which advocates more in vivo

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evaluations or human trials.

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KEYWORDS: Formononetin; Ononin; Bioavailability; Permeability; Caco-2;

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Ussing chamber


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INTRODUCTION

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Formononetin is a dietary isoflavone and a potent phytoestrogen widely present in

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legumes such as soy, kidney beans and navy beans as free aglycone or the substituted

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glucoside conjugate ononin.1-3 Formononetin and ononin (Figure 1) are also major

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types of flavonoes found in various Chinese herbal medicines, such as red clover

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(Trifolium pratense L.), glycyrrhizae radix (the root of licorice) and astragali radix

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(the root of Astragalus membranaceus var. mongholicus or A. membranaceus).3-6

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Dietary intake of isoflavones has been associated with alleviation of osteoporosis,

26

climacteric symptoms and vascular disease,7-9 as well as lower risk of breast cancer

27

and prostate cancer.10-11 Notably, formononetin and ononin have been reported to

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exhibit remarkable anti-oxidant, anti-inflammatory and hypolipidemic effects in

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various in vitro and animal models.12-16 Recent reports have demonstrated that

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formononetin had neuroprotective effect in rat models of ischemia/reperfusion injury

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and traumatic brain injury,17-18 protected C57BL/6 mice from high-fat diet-induced

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obesity and bone loss through inhibition of adipogenesis,19 and inhibited angiogenesis

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and tumor growth in breast cancer xenograft models via inhibition of fibroblast

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growth factor receptor 2 (FGFR2).20

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In contrast to the widely-explored pharmacological actions, the studies on the in



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vivo fate of formononetin and ononin are limited. Several researchers have

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investigated the pharmacokinetics of formononetin and ononin in rats after oral

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administration of herbal extracts of Astragali radix and red clover, or Chinese herbal

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formulas

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bioavailability of these two individual isoflavones remain to be addressed. On the

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basis of current knowledge on other isoflavones, low gut permeability and extensive

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metabolism often lead to poor bioavailability. Previously, it was reported that both

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hepatic microsomal enzymes and gut microbiota played key roles in extensive

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metabolism of formononetin and ononin.24-25 However, to date, there is little

45

information available on gut permeability of these two isoflavones.26 Moreover, it is

46

unclear that whether transporters have been involved in their absorption.

containing

these

herbs.21-23

However,

the

pharmacokinetics

and

47

Therefore, the present study aims to investigate the pharmacokinetics and

48

bioavailability of formononetin and ononin in rats, and to monitor their gut

49

permeability properties in Ussing chamber and Caco-2 cell models.

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MATERIALS AND METHODS

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Materials and Reagents. Formononetin, ononin and calycosin-7-O-glucoside

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(internal standard, IS) were bought from Solarbio (Beijing, China). Sodium carboxyl

53

methyl cellulose (CMC-Na) was purchased from Tianjin Chemical Reagent Company

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(Tianjin, China). Methanol and acetonitrile of HPLC grade were obtained from Merck

55

(Darmstadt, Germany). Deionized water was purified by a Milli-Q purification system


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(Millipore; Bedford, MA). Heparin, dimethyl sulphoxide (DMSO), Hanks’ balanced

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salt solution (HBSS), tween 80, HEPES, verapamil, and pravastatin was purchased

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from Sigma-Aldrich (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM),

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fetal bovine serum (FBS) and nonessential amino acids were supplied by Gibco

60

(Grand Island, NY). Caco-2 cells were obtained from the American Type Culture

61

Collection (Rockville, MD).

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Pharmacokinetic Study. The care of animals and all experimental procedures

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were approved by the Committee on Use and Care of Animals of China Academy of

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Chinese Medical Sciences. Male Sprague-Dawley (SD) rats (210-230 g) supplied by

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the Experimental Animal Centre, China Academy of Chinese Medical Sciences

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(Beijing, China) were maintained under controlled conditions of temperature (22-24

67

o

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water ad libitum. Right jugular vein cannulation was performed on anesthetized rats

69

on the day before isoflavones administration. The rats were housed individually in

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metabolic cage, and allowed to recover and fasted overnight with free access to water.

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Right jugular vein was cannulated with polyethylene tube for blood sampling.

C), humidity (55-60 %), and a light/dark cycle of 12/12 h with access to food and

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Rats were randomly grouped into four groups (6 rats per group). Formononetin or

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ononin was dissolved in 0.5% CMC-Na or in 5% tween 80 for oral and intravenous

74

administration, respectively. As tween 80 has been reported to affect transporter

75

activity in gut,27 it is not used as a solvent for oral administration. Group 1 and group

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2 rats were orally treated formononetin or ononin (20 mg/kg). Blood (about 0.2 mL)



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were collected in heparinized tube at 0, 5, 15, 30 min, 1, 2, 4, 6, 8 h after drug

78

administration. Group 3 and 4 rats received intravenously formononetin or ononin (4

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mg/kg) via tail vein. Blood (about 0.2 mL) were collected in heparinized tube at 0, 2,

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5, 15, 30 min, 1, 2, 4, 6, 8 h after drug administration. After each sampling, an equal

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volume of heparinized normal saline was given to rats immediately for compensation

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of blood withdrawal. Blood were centrifuged at 1500 g for 10 min to obtain plasma

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and stored at -20 oC until analysis.

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For sample preparation, 80 µL ice-cold methanol containing 100 nM IS was added

85

to 40 µL plasma sample, which was then vortexed for 1 min and centrifuged at 15,000

86

g for 10 min at 4 oC. The supernatant was used for LC-MS detection.

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Ussing Chamber. The Ussing chamber system, equipped with intestinal tissues

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from rats, was prepared as previously reported.28-29 In brief, rat gastrointestinal

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segments, including stomach, duodenum, jejunum, ileum, cecum and colon, after

90

removing the muscle layer, were vertically mounted in Ussing chambers. The

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transport buffer of Krebs-Ringer solution (5 mL) was added into both apical and basal

92

sides, which was maintained at 37 oC and continuously supplied with 95% O2/5%

93

CO2. The available area for drug transportation was 0.5 cm2. The transepithelial

94

electrical resistance (TEER) was measured before and after the study. A decrease of

95

TEER (>15%) indicates the leakage of intestinal tissues.30-31 After a 15-min

96

equilibration, transport buffer in the apical side was replaced with pre-warmed buffer

97

containing 10 µM formononetin or ononin. Samples (100 µL) were collected from the


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basal side at 0, 15, 30, 45, 60 and 120 min. At the end of sampling, 100 µL solution

99

was collected from the apical side for analysis to calculate recovery. All samples were

100

stored at -20 oC until analysis.

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For sample preparation, 100 µL samples were mixed with 300 µL ice-cold

102

methanol containing 50 nM IS, followed by vortexed for 1 min and centrifuged at

103

15,000 g for 10 min at 4 oC. The supernatant was used for LC-MS detection.

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Caco-2 Monolayer Transport Assay. Caco-2 cells were cultured in a DMEM

105

medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids

106

and 1% penicillin and streptomycin at 37 oC in 5% CO2, and were seeded into 6-well

107

transwells (0.4 µM, polyester membrane, 24 mm insert; Corning, NY) at 2 × 105

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cell/mL for 22 days. The integrity of the monolayer was verified by measuring the

109

apparent permeability coefficients (Papp) values of the paracellular marker atenolol

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and the transcellular marker propranolol, and the TEER using an epithelial tissue

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voltohmmeter (World Precision Instrments, Sarasota, FL). Monolayers with TEER >

112

300 Ω‧cm2 (subtracting the background value of a transwell) before and after

113

transport assay and appropriate Papp values for atenolol (around 10-7 cm/s) and

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propranolol (around 10-5 cm/s) were used for transport study.

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To see whether transporters were involved in the absorption of formononetin and

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ononin, the bidirectional transport assays with or without verapamil (a specific P-gp

117

inhibitor) or pravastatin (a specific MRP2 inhibitor) were performed. Transport



118

studies were conducted at 37 oC, in HBSS containing 25 mM HEPES. Prior to study,

119

medium from both sides of the monolayers was removed, and replaced with HBSS

120

alone, or HBSS containing verapamil (50 µM) or pravastatin (1 mM). Following a

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15-min incubation, formononetin or ononin (10 µM) was added into the basolateral

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side for the basolateral to apical (B to A) transport study or apical side for the apical

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to basolateral (A to B) transport study. At designated times (0, 15, 30, 60, 90 min),

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100 µL sample was collected from the receiver compartment, and then 100 µL HBSS

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solution was added immediately to maintain a constant volume. At the end of

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sampling, 100 µL solution was collected from the donor compartment for LC-MS

127

analysis to calculate recovery. Samples were stored at -20 °C until LC-MS analysis.

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Sample preparation was conducted as described in Ussing chamber study.

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LC-MS Analysis. Quantitative analysis was conducted on an Agilent UHPLC

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1290 system equipped with a vacuum degasser, a binary pump, an autosampler and a

131

6460 triple quadruple mass spectrometry.32 A ZORBAX Eclipse Plus C18 column (2.1

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× 100 mm, 1.7 µm, Agilent Technologies) was used for chromatographic separation.

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The column was maintained at 35 °C. The mobile phases were acetonitrile containing

134

0.1% formic acid (A) and water containing 0.1 % formic acid (B). The elution rate

135

was 0.3 mL/min and the gradient program was as follows: 0-1 min 15% B; 1-4 min

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15-60% B; 4-5 min 60-90% B; 5-6 min 90% B. The eluate from column for the first 1

137

min was directed to the waste. The injection volume is 10 µL for both formononetin

138

and ononin.


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The mass spectrometer was operated in positive ion mode with an electrospray

140

ionization (ESI) interface. Quantitation was performed by multiple reactions

141

monitoring (MRM). In the positive mode, the MS/MS parameters were as follows:

142

capillary voltage 3500 V; gas temperature 280 oC; sheath gas heater 350 oC; sheath

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gas flow 11 L/min; gas flow 5 L/min. The precursor to product ion pairs for

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monitoring formononetin, ononin and IS in the MRM mode were m/z 269→197, m/z

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431→269, and m/z 447→285, respectively. The fragmentor was set at 135 V, while

146

collision energies were 45 V for formononetin and 20 V for ononin and IS.

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Data Analysis. Oral bioavailability (F) was calculated as: F = (AUCp.o.*Dosei.v.)/

148

(AUCi.v.*Dosep.o.)*100%. Papp value of formononetin and ononin for the bidirectional

149

transport in Caco-2 monolayers and in Ussing chambers was calculated as:33 Papp =

150

(dC/dT*V)/A/C0, where dC/dT is the initial slope of the plot of cumulative

151

concentrations versus time (µM/s); V is the volume of solution in receiver chamber,

152

which is 0.5 mL for the apical side and 1.5 mL for the basolateral side in Caco-2

153

model, and 5 mL in the Ussing chamber model; A is the surface area of the monolayer,

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which was 1.12 cm2 for the 12-well Transwell plate and 0.5 cm2 for the Ussing

155

chamber; C0 is the initial concentration in donor site (µmol/mL). The Papp values are

156

commonly used for the prediction of absorption of orally administered drugs. It is

157

suggested that drugs with Papp < 1 × 10-6 cm/s, Papp = (1-10) × 10-6 cm/s, and Papp > 10

158

× 10-6 cm/s usually have poor, moderate and good absorptions, respectively.34 An

159

efflux ratio (Papp (B to A)/ Papp (A to B)) larger than 1.5 was used for determining whether



160

efflux transporters were involved in the drug transport in Caco-2 model.

161

All data are expressed as mean ± standard deviation (SD). Statistical difference

162

was analyzed by GraphPad Prism 7.0 using unpaired student’s t test (for comparison

163

between two groups) or one-way ANOVA with a post hoc Tukey test (for comparison

164

among three or more groups). A p < 0.05 was considered significant.

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RESULTS AND DISCUSSION

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Pharmacokinetics and Bioavailability of Formononetin and Ononin.

167

Determination of the concentration of formononetin and ononin in rat plasma was

168

performed using LC-MS method. As shown in Table 1, the calibration curves of

169

formononetin and ononin provided good linearity (R2 > 0.99) within the concentration

170

ranges tested. The limits of quantification (LOQs) were 0.5 and 1.0 nM for

171

formononetin and ononin, respectively. The results (Supplementary Table 1) for

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precision (RSD, from 0.7% to 6.9%), accuracy (standard error, from -8.5% to 9%) and

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extraction recovery (from 89.2 ± 8.2% to 100.7 ± 6.9%) indicated the reliability of

174

methods for determination of formononetin and ononin.

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The plasma concentration versus time curves of formononetin and ononin after

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oral or intravenous administration are shown in Figure 2, with pharmacokinetic

177

parameters displayed in Table 2. The oral dosages of formononetin and ononin were

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set at 20 mg/kg based on previous pharmacological studies.35-36 After an oral

179

administration at 20 mg/kg, formononetin was rapidly absorbed and peaked at 30 min,


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which was followed by a quick elimination phase with a half-life of 2.1 h, whereas

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ononin showed a double peak phenomenon in the plasma concentration profile, which

182

were characterized by Tmax1 (or Cmax1) and Tmax2 (or Cmax2) (Table 2). The first peak

183

was observed at around 30 min, indicating a rapid absorption of ononin, then

184

experienced a quick decline and a subsequently small rise that peaked at 4 h after oral

185

administration (Figure 2). The rapid absorption of formononetin and ononin was

186

observed similar to other reported isoflavones such as genistein, daidzein, daidzin and

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puerarin with Tmax of 0.25-1 h.37-39 Furthermore, in consistent with previous

188

studies,40-41 the double peak phenomenon of ononin has been reported after oral

189

administration of other herbal extracts, which was further discussed in the next

190

section after the gut permeability study.

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It should be noted that after an oral administration of ononin, formononetin was

192

detected as a metabolite in rat plasma, and displayed a double peak phenomenon. The

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first peak of formononetin was observed at 1 h post ononin dosing, with a Cmax1 of

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35.2 ± 10.1 nM, and the second peak was seen at around 6 h, with a larger Cmax2 of

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76.5 ± 12.5 nM. It has been demonstrated that ononin could be biotranformed into

196

formononetin via colonic microbiota-mediated deglycosylation.25 Moreover, there is

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evidence that, compared to colonic microbiota, isoflavone glycosides such as

198

quercetin-3-glucoside, genistein-7-glucoside and daidzein-7-glucoside can be

199

hydrolyzed, to a lesser extent, by the lactase phlorizin hydrolase (LPH), an enzyme

200

found on the brush border of the mammalian small intestine.42 Although not being



201

proved, ononin may be deglycosylated by LPH in small intestine in a way similar to

202

other isoflavone glycosides. Therefore, hydrolysis of ononin at double sites (both

203

small and large intestines) might be responsible for the double peak phenomenon of

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formononetin after oral administration of ononin.

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Notably, after giving a same oral dose, the systemic exposure of formononetin

206

(AUC0-8 h, 713.4 ± 46.2 nM*h) was about 4.5-fold higher than that of ononin (AUC0-8

207

h,

208

4 mg/kg, formononetin and ononin displayed similar biphasic plasma concentration

209

profiles (Figure 2), with the AUC0-∞ of 652.3 ± 89.1 and 439.5 ± 54.1 nM*h,

210

respectively (Table 2). Based on the results, formononetin was moderately

211

bioavailable in rats with an absolute bioavailability of 21.8%, while ononin had a

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relatively low bioavailability of 7.3%. Since ononin was rapidly biotransformed in gut

213

to generate formononetin and was further metabolized,43 it is not a surprise that

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ononin is less bioavailable than formononetin. Besides, the aglycones are generally

215

more liposoluble than their corresponding glycosides, and hence easier to transport

216

across the gut. However, when the formononetin as an active metabolite of ononin

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was taken into consideration for calculation, the bioavailability of ononin reached

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21.7%, which was similar to that of formononetin. That is to say, ononin is more

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bioavailable in vivo than perceived.

160.2 ± 39.1 nM*h). On the other hand, following an intravenous administration at

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Most human studies of isoflavones and flavonoids have been unsuccessful, which

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is mainly due to the very poor oral bioavailability.44-45 Isoflavones and flavonoids


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such as chrysin, quercetin and resveratrol usually contain several free hydroxyl groups

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and facilitate rapid intestinal/hepatic phase II metabolism by glucuronidation and/or

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sulfation for excretion.46-48 It is reported that methylated flavones have increased

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metabolic stability and improved intestinal transport.49-50 Thomas Walle et al. showed

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that 5,7-dimethoxyflavone and 5,7,4’-trimethoxyflavone had higher bioavailability

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and tissue distribution in rat in contrast to the unmethylated chrysin and apigenin.51

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Formononetin is a naturally occurring methylated isoflavone. Compared with other

229

hydroxylated ones such as biochanin A, formononetin has been demonstrated to

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display slower glucuronidation in intestinal and hepatic microsomes and higher gut

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permeability.52 The moderately bioavailable formononetin might have benefited from

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its methylation status in its structure.

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Absorption of Formononetin and Ononin at Different Gastrointestinal

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Segments. As one of the key limiting steps for entering circulation system, how gut

235

permeability contribute to the bioavailability differences of the parent formononetin

236

and ononin is further evaluated using a Ussing chamber model. Samples were

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determined using LC-MS method, and the calibration curves and quantification limits

238

are shown in Table 1. The Papp values for absorption of formononetin and ononin at

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different intestinal segments are displayed in supplementary Table 2 and Figure 3. As

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shown in supplementary Table 2, the recoveries of the two compounds in the assay

241

were generally above 80% (except formononetin using duodenum segment and

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ononin using jejunum segment with mean recovery of 79%), indicating that the Papp



243

values were within acceptable accuracy.53-54 We chose 10 µM of formononetin and

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ononin for the Ussing chamber study, because initial results showed su‧cient

245

isoflavones at the donor and receiver sides to allow accurate measurement.

246

The results (Figure 3 and supplementary Table 2) showed that both formononetin

247

and ononin were better absorbed in the large intestine segments (cecum and colon),

248

with Papp values of (5.98-6.10) × 10-6 cm/s (formononetin) and (1.16-1.91) × 10-6 cm/s

249

(ononin), in comparison to that in the small intestine segments (duodenum, jejunum

250

and ileum), with Papp values of (1.52-2.45) × 10-6 cm/s (formononetin) and (0.10-0.25)

251

× 10-6 cm/s (ononin). Similar result has also been reported on biochanin A.52 This

252

might be explained by the site-specific distribution of these isoflavones in the gut tract.

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It was found that genistein had a high tissue distribution in gut, with a highest

254

residence in cecum at specific time point after administration in rats.39 In this study,

255

the site-specific absorption behavior was more obvious for ononin, which supported

256

the finding that ononin showed a double peak phenomenon in its plasma

257

concentration profile. It is anticipated that, after oral administration, ononin had a

258

small absorption in small intestine, and most parts were absorbed in the large intestine.

259

The fact that double peak phenomenon did not occur after intravenous administration

260

of ononin indicated that it was not due to enterohepatic cycling. Thus, it is highly

261

possible that double-site absorption leads to the double peak phenomenon of oral

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ononin. On the other hand, although formononetin showed a better permeability in the

263

large intestine segments, it had a moderate absorption (Papp > 1 × 10-6 cm/s) in the


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small intestines segments. Due to the much longer length of small intestine than large

265

intestine, formononetin should have a longer residence in the small intestine. In this

266

regard, small intestine may be the main site responsible for formononetin absorption,

267

and this might be the main reason for why formononetin did not display double peak

268

phenomenon in its plasma concentration profile.

269

Interestingly, ononin could not transport across stomach tissue, whereas

270

formononetin showed a poor absorption with a Papp value of (0.20 ± 0.04) × 10-6 cm/s.

271

This is in consistent with previous report that certain isoflavonoid aglycones such as

272

daidzein and genistein but not their glycosides can be absorbed in stomach.55 It was

273

found that after only 3 minutes of administration, daidzein and genistein and their

274

metabolites were detected in the rat plasma. Compared to the isoflavone aglycones,

275

their glucosides, daidzin and genistin, were found in the plasma with only a few

276

minutes’ delay. The delay was due to the time required by the administered glucosides

277

to pass across stomach before reaching the small intestine where they were absorbed.

278

Therefore, both isoflavones and the glucosides are rapidly absorbed in rats regardless

279

of their permeability difference in stomach. This supported that both formononetin

280

and ononin had a similar Tmax at around 30 min.

281

It should be noted that, in all segments of intestines, the Papp values of

282

formononetin were significantly higher than that of ononin (Figure 3), indicating a

283

better permeability of the more liposoluble formononetin. Importantly, the relatively

284

low permeability (Papp < 2 × 10-6 cm/s) of ononin in gut is proposed as an alternative



285

contributor for its poor bioavailability.

286

Transport of Formononetin and Ononin across Caco-2 Cell Monolayer.

287

Caco-2 model is widely applied to predict drug transport via different ways, including

288

the passive diffusion and paracellular routes, the transporters-mediated route and

289

transcytosis, across the human intestinal epithelium.56 In vitro permeability of

290

formononetin and ononin was carried out in this model. In the present permeability

291

assay, recoveries of formononetin and ononin were above 77% (Table 3), suggesting

292

an acceptable accuracy for the Papp values.57-58 At 10 µM, both formononetin and

293

ononin did not cause cytotoxicity on Caco-2 cells for 24 h incubation, suggesting that

294

formononetin and ononin at the selected concentration would not affect the integrity

295

of Caco-2 cell monolayer.

296

The Papp (A to B) values for formononetin and ononin across Caco-2 cell monolayers

297

are summarized in Table 3. The results showed a moderate absorption of

298

formononetin with a Papp

299

absorption of ononin with a Papp (A to B) value of (3.58 ± 0.25) ×10-7 cm/s, which was 1

300

order of magnitude lower than that of formononetin. These data were in consistent

301

with the Ussing chamber results, which again indicated that the better permeability of

302

formononetin might contribute to its higher bioavailability over ononin. High

303

permeability (> 5 ×10-6 cm/s) of isoflavones in Caco-2 cell model has been reported

304

previously regardless of the methylated status in structures.26, 59 Based on previous

(A to B)

value of (5.14 ± 0.22) ×10-6 cm/s, and a poor


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reports59 and the present study, the more liposoluble isoflavones generally have better

306

permeability over others, especially the unmethylated ones or the glycosides.

307

However, higher gut permeability is not always correlated with higher bioavailability.

308

Genistein was reported to less permeable than the more liposoluble daidzein in gut,26

309

whereas it was found that genistein was more bioavailable than daidzein.60

310

To investigate whether efflux transporters were involved in the absorption,

311

bi-directional transport assay was performed. The bilateral Papp values are shown in

312

Table 3. The efflux ratio (Papp (B to A)/Papp (A to B)) of formononetin was within the range

313

of 1.0-1.5, indicating that there was no significant difference between permeability in

314

A to B direction and that in B to A direction, and transportation of formononetin

315

across intestinal epithelial cells was mainly through passive diffusion.

316

On the other hand, the transportation of ononin from A to B direction was

317

significantly lower than that from B to A direction, with an efflux ration of 1.656,

318

suggesting that ononin might be substrate of efflux transporters. The addition of

319

verapamil (a specific P-gp inhibitor) did not affect the bilateral transport of ononin,

320

whereas pravastatin (a specific MRP2 inhibitor) significantly inhibited the preferential

321

transportation of ononin from B to A direction. The results indicated that ononin was a

322

substrate of MRP2 but not P-gp, and its low bioavailability might be partially

323

attributed to MRP2-mediated efflux during absorption. This was in consistent with

324

other report where they found that transport of the flavonoid glycoside

325

quercetin-4’-β-glucoside was specifically through MRP2 but not P-gp.61 MRP2 ACS Paragon Plus Environment


326

governs the transportation of its substrates across the intestinal wall. Previous studies

327

have demonstrated that MRP2 mostly expresses at the apical side of epithelia in small

328

intestine including duodenum, jejunum and ileum, with almost no expression in the

329

entire colon segment.62 The lower permeability of ononin in the small intestine

330

compared to the large intestine might be due to the differential efflux activity of

331

MRP2 in these intestinal segments.

332

In summary, the present study revealed the pharmacokinetic properties of

333

formononetin and ononin after oral and intravenous administration in rats.

334

Formononetin had a bioavailability of 21.8%, and was absorbed in all gastrointestinal

335

segments with varied permeability. In Caco-2 model, formononetin showed moderate

336

absorption via passive diffusion. Meanwhile, ononin displayed a bioavailability of

337

7.3%. When formononetin as an active metabolite of ononin was taken into

338

consideration, the bioavailability of ononin reached 21.7%. Ononin was poorly

339

absorbed in all intestine segments of rats, and had a poor permeability in Caco-2

340

monolayer, which was identified as a substrate of MRP2 but not P-gp. These findings

341

facilitated the understanding of the in vivo pharmacological actions of formononetin

342

and ononin from a pharmacokinetic view, and also implicated that both formononetin

343

and ononin, due to their moderate bioavailability, might have a potential for more in

344

vivo evaluations or even human trials.

345

ABBREVIATIONS


Page 18 of 32

Page 19 of 32


346

DMEM, Dulbecco’s modified Eagle’s medium; FGFR2, Fibroblast growth factor

347

receptor 2; LPH, lactase phlorizin hydrolase; MRM, multiple reaction monitoring;

348

MRP2, multidrug resistance protein 2; Papp, apparent permeability coefficient; TEER,

349

transepithelial electrical resistance.

350

SUPPORTING INFORMATION

351

Table 1 about the validation of LC-MS method for determining formononetin and

352

ononin in rat plasma in terms of precision, accuracy and recovery, and table 2 about

353

the Papp values and recoveries of formononetin and ononin at different gastrointestinal

354

segments in Ussing chambers.

355

ACKNOWLEDGEMENT OF FINANCIAL SUPPORT

356

This work was supported by the Funding of Hong Kong, Macao & Tianwan Science

357

and Technology Cooperation Project (2015DFM30030), and the Fundamental

358

Research Funds for the Central Public Welfare Research Institutes (ZZ10-007).

359 360

REFERENCES

361 362 363 364 365 366 367 368 369 370 371 372 373 374

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557


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Figure Captions: Figure 1. Structures of the isoflavone formononetin and its glycoside ononin. Figure 2. Plasma concentration versus time curves of formononetin (A and B) and/or ononin (C and D) after oral or intravenous administration in rats. Rats (n = 6 in each group) were treated with formononetin or ononin via p.o. (20 mg/kg) or i.v. (4 mg/kg). Blood were obtained through cannulated right jugular at the designated time points. Plasma concentrations of formononetin or ononin were analyzed by LC-MS method. Data are represented as mean ± SD (n = 6). Figure 3. In vitro absorption of formononetin (A) and ononin (B) at different gastrointestinal segments in Ussing chamber model. Gastrointestinal segments, including stomach, duodenum, jejunum, ileum, cecum and colon, were mounted in the Ussing chambers after removal of the muscle layer. Transfer buffer containing 10 µM formononetin or ononin was added in the apical side. Apparent permeability coefficients (Papp) values for formononetin (A) and ononin (B) after 120-min transport are shown. Statistical analysis was performed using Graphpad Prism 7.0. Data are obtained from three independent experiments and are displayed as mean ± SD (n = 3).



Page 26 of 32

Table 1. Calibration and Limits of Quantification (LOQ) for Formononetin and Ononin in Different Matrices. Compounds

Matrices

Calibration curvesa

Ranges (nM)

R2

LOQ (nM)

Formononetin

Plasma HBSS Krebs-Ringer buffer Plasma HBSS Krebs-Ringer buffer

Y = 9.501*10-3X + 4.301*10-4 Y = 0.0338X - 3.088*10-4 Y = 0.0549X + 0.00101

5-2000 1-500 1-500

0.991 0.989 0.994

0.5 0.2 0.2

Y = 0.0169X + 8.09857*10-4 Y = 0.0750X - 0.00107 Y = 0.0969X + 8.090*10-4

5-2000 1-400 1-400

0.990 0.990 0.997

1.0 0.5 1.0

Ononin

a

Y represents peak area ration of analyte and IS while X is the concentration of analyte. The injection volume is 10 µL for formononetin and ononin. The calibration curve of each analyte in different matrices was constructed by plotting peak area ratio of analyte and IS versus spiked concentration. The LOQs were determined using the signal to noise (S/N) ratio of 10.


Page 27 of 32


Table 2 Pharmacokinetic Parameters of Formononetin and Ononin after an Oral or Intravenous Administration. Formononetin Ononin Parameters 20 mg/kg, 20 mg/kg, p.o. 4 mg/kg, 4 mg/kg, i.v. p.o. i.v. Ononin Formononetin 1302.8 ± Cmax (nM) 302.1 ± (1) 74.6 ± (1) 35.2 ± 10.1 1142.2 ± 129.1 35.9 26.1*** (2) 76.5 ± 12.5 103.2 (2) 34.0 ± 8.4 AUC0-8 h 317.2 ± 49.1 439.5 ± 713.4 ± 652.3 ± 89.1 160.2 ± 39.1### 54.1† (nM*h) 46.2 AUC0-∞ 757.7 ± 689.5 ± 92.1 173.3 ± 43.1§§§ 463.8 ± (nM*h) 48.2 60.1‡ Tmax (h) 0.5 ± 0.0 (1) 0.5 ± 0.0 (1) 1.0 ± 0.0 (2) 4.0 ± 0.0 (2) 5.7 ± 0.0 t1/2 (h)

2.10 ± 0.28

2.23 ± 0.6

1.82 ± 0.56

-

1.92 ± 0.8

Vz (L/kg) CL (L/h/kg) F%

-a -

13.9 ± 1.0 4.3 ± 0.8

-

-

18.4 ± 1.5 6.6 ± 0.9

21.8%

-

7.3% (21.7%b)

-

-

a

“-”, not applicable.

b

The calculation has included both AUC of formononetin and ononin after oral ononin administration.

Data are expressed as mean ± SD (n=6). ***p < 0.001, ###p < 0.001, or §§§p < 0.001, compared with the Cmax, AUC0-8 h, and AUC0-∞ of p.o. formononetin, respectively;

†

p < 0.05, or

‡

p < 0.05, compared

with the AUC0-8 h, and AUC0-∞ of i.v. formononetin, respectively. Cmax, peak plasma concentration after drug administration; AUC, area under the curve; Tmax, time at peak concentration; t1/2, half life; Vz, volume of distribution; CL, clearance; F, bioavailability.



Page 28 of 32

Table 3 Apparent Permeability Coefficients (Papp) Values and Efflux Ratio of Formononetin and Ononin Determined in Caco-2 Model. Recoveryb

Papp (×10-6 cm/s) Efflux ratioa

Isoflavones

Formononetin Ononin

A to B

B to A

A to B

B to A

5.143 ± 0.220

5.515 ± 0.139

1.174

77.8 ± 4.7 95.1 ± 5.3

0.358 ± 0.025*** 0.593 ± 0.127#

1.656

87.4 ± 5.7 87.9 ± 10.8

Ononin+Verapamil

0.349 ± 0.151

0.661 ± 0.102†

1.893

81.8 ± 9.1 86.2 ± 1.3

Ononin+Pravastatin

0.443 ± 0.117

0.417 ± 0.390

0.941

78.9 ± 4.2 90.0 ± 13.3

a

Efflux Ratio, Papp (B to A)/Papp (A to B), where Papp (B to A) is the average of the permeability coefficient

from basolateral (B) to apical (A); Papp (A to B) is the average of the permeability coefficient from A to B. b

Recovery was calculated as the ratio of total amount of drugs detected in A and B sides after

90-min transport study and the nominated amounts initially added. Verapamil (a specific P-gp inhibitor) and pravastatin (a specific MRP2 inhibitor) were used to investigate whether transporters were involved in the bidirectional transportation of isoflavones. Data are expressed as mean ± SD (n = 3). ***p < 0.001, compared with Papp (A to B) of formononetin; #p < 0.05, compared with Papp (A to B) of ononin; †p < 0.05, compared with Papp (A to B) of ononin in the presence of verapamil. Papp, apparent permeability coefficients.


Page 29 of 32


Figure 1.



Figure 2.


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Figure 3.



Graphic for table of contents


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Pharmacokinetics and Bioavailability of the Isoflavones Formononetin

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